1. Field of the Invention
This invention relates to the analysis of a body fluid, and more particularly relates to a method for the assessment of a bacteriuria by the rapid and simultaneous detection and quantitation of microorganisms, leukocytes and squamous epithelial cells in urine.
2. Description of the Prior Art
Bacteriuria is defined conventionally as the presence of significant numbers of pathogenic microorganisms in the urine, and can arise from colonization of the urine or from urinary tract infection. Colonization in the urinary tract is defined conventionally as replication of organisms normally found in the urine which is not accompanied by invasion of adjacent epithelial and subepithelial tissues. Urinary tract infection is defined conventionally as bacteriuria accompanied by clinical, histologic, or immunologic evidence of host injury.
Urine specimens represent a large portion of the samples received by a clinical diagnostic laboratory. In collection of urine specimens, care must be taken to avoid contamination. Contamination is defined as the inadvertent entry into the collecting vessel of microorganisms normally found in tissues surrounding the urinary tract, and most often is a problem in specimen collection from female patients.
Current methodology used for the detection and quantitation of bacteriuria involves culturing of the microorganisms in the specimen. In these methods, aliquots of urine (usually 1-10 ul) are plated onto various agar type surfaces, cultured for 18-24 hours, and the colonies are counted. In culturing methods, the appearance of colonies of three or more types of organisms suggests specimen contamination.
Screening methods which do not rely on organism growth have been introduced for the rapid detection of bacteriuria. In general, the purpose of screening is to identify and eliminate those urine specimens, generally referred to as negatives, that do not contain significant numbers of microorganisms. Screening, however, is complicated by the fact that urine is not, even in healthy individuals, a sterile fluid, and there has been considerable debate about what urinary microorganism levels indicate infection and what levels are normal. Reviews of this subject in light of conventional methods of urine culture have been presented by Stamm and by Pollock (Am. J. Medicine, Proceedings of a Symposium on Body Fluids and Infectious Diseases, p. 53 and 79, July 28, 1983). Most investigators now consider significant bacteriuria to be defined as the presence of pathogenic microorganisms in a urine specimen at levels of 1.times.10.sup.4 -1.times.10.sup.5 colony forming units (cfu)/ml and greater. Recent studies, however, by Stamm et al., N. Engl. J. Med. 307, 463 (1982), have shown that defining urinary tract infection as 1.times.10.sup.5 cfu/ml resulted in a sensitivity of only about 50% for symptomatic patients.
The Gram stained smear of uncentrifuged urine specimens is occasionally used as a conventional screening procedure for bacteriuria with a high accuracy for predicting negatives. However, the method is highly labor intensive and is not deemed practical when large numbers of samples are received daily. Recently, other non-growth methods have been developed. The BAC-T-SCREEN.TM. system, Marion Scientific, Kansas City, Mo., is based on filtration and staining and identifies positive urine specimens which warrant further study via plating. The LUMAC.TM. system, (3M Co., Minneapolis, Minn.) is based on detection of bacterial adenosine triphosphate and identifies positive specimens in 30 minutes.
For those urine specimens for which either a culturing or screening procedure indicates a significant bacteriuria, differentiation between specimen contamination, colonization and infection is generally carried out. If the bacteriuria is accompanied by large numbers of squamous epithelial cells, specimen contamination is indicated as a possible cause of the bacteriuria. In general, a threshold level of 1.times.10.sup.4 squamous epithelial cells per ml of urine specimen is considered to be indicative of contamination. In the art, epithelial cell counts are conventionally carried out during urinalysis in a hemocytometer or by staining a smear of a centrifuged urine specimen.
In the absence of specimen contamination, differentiation of colonization and infection is generally done by measuring the level of pyuria (the presence of leukocytes in the urine). Leukocytes are normally present in urine, and careful studies have established the threshold limit for pathological pyuria as about 1.times.10.sup.4 leukocytes/ml of uncentrifuged urine. Pyuria correlates closely with acute symptomatic infection, and its presence may warrant further study of urine specimens in which microorganism counts are less than accepted threshold levels.
Several methods have been developed for assessment of pyuria. These include measurement of leukocyte counts obtained in a hemocytometer or in the high power field of a microscope on spun or unspun urine samples. These methods all have high variability and require too much time and effort for routine clinical use. A currently available dip-and-read test strip based on leukocyte esterase (CHEMSTRIP.TM., Biodynamics, Indianapolis, IN.) changes color when dipped into urine containing more than 1.times.10.sup.5 leukocytes/ml.
All currently available systems for detection of bacteriuria suffer from disadvantages. Growth based methods have the disadvantages of the time required for culturing and the cost of materials and labor used on samples which ultimately prove negative. Methods in which urine specimens are designated as negatives on the basis of microorganism counts alone (Gram stain, LUMAC.TM. and BAC-T-SCREEN.TM.) run the risk of missing positive urine specimens from true urinary tract infections in which the microorganism counts are less than the accepted threshold value of significant bacteriuria (1.times.10.sup.5 cfu/ml). The Gram stain, LUMAC.TM. and BAC-T-SCREEN.TM. systems, although providing short analysis time, require substantial hands-on time per sample. CHEMSTRIP.TM. although quick and inexpensive to use, has the disadvantage of missing some positives. Further, none of the prior art methods assay for all three cell types (microorgansims, leukocytes and squamous epithelial cells) which may be involved in bacteriuria. Current methodology requires separate assays, and these are often carried out in different laboratories. In current practice, microorganism determinations are generally handled in a hospital microbiology laboratory while leukocyte and squamous epithelial cell determinations are handled in the hospital urinalysis laboratory. Thus, there is a need for a better method for the rapid detection of bacteriuria and differentiation of the three cell types often associated with bacteriuria.